The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

Yang Zhang, Adnan Halim, Yoshiki Narimatsu, Hiren Jitendra Joshi, Catharina Steentoft, Katrine Ter-Borch Gram Schjoldager, Morten Alder Schulz, Natalie R. Sealover, Kevin J. Kayser, Eric Paul Bennett, Steven B. Levery, Sergey Y. Vakhrushev, Henrik Clausen

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The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (
Original languageEnglish
JournalMolecular and Cellular Proteomics
Issue number12
Pages (from-to)3224-3235
Publication statusPublished - 2014


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